DESCRIPTION The overall objective is to understand the molecular basis for the production of more catalytically efficient cytochrome P450 enzymes. The P450 family of proteins metabolized a wide spectrum of drugs and environmental chemicals as well as endogenous substrates like fatty acids and steroids. Eukaryotic microsomal cytochrome P450s have catalytic turnover rates >1000 times slower than prokaryotic P450s, but they metabolize a wider variety of compounds than prokaryotic P450s. Does broad substrate specificity come at the expense of catalytic efficiency, or have other biological requirements limited the catalytic rates of microsomal enzymes? Can microsomal P450s be evolved with higher catalytic rates without compromising substrate specificity? These questions will be addressed by: 1) evolving microsomal P450 2E1 genes, in vitro, substrate specificity? These questions will be addressed by: 1) evolving microsomal P450 2E1 genes, in vitro, and identifying enzymes with increased catalytic activity, 2) examining changes in substrate affinities and kinetic properties of these enzymes, and 3) further improving catalytic activity by additional rounds of evolution. Experiments will include the generation of mutant libraries by in vitro homologous recombination and/or error-prone PCR, high-throughput screening to identify functional mutants, and sequence and kinetic analysis of proteins to delineate underlying mechanisms contributing to altered activities. The proposed work will contribute to our understanding of the unique catalytic activities of P450s as well as demonstrate the feasibility of evolving P450s with altered functions. Engineering P450s with specific functions have desirable applications in medicine and the environment.